The quantitative determination of hemoglobin in blood is one of the most frequently performed analyses in clinical chemistry. For this purpose various methods have been developed in the course of this century, which differ considerably from each other with respect to accuracy, reliability and reproducibility [cf. L. Hallmann, Klinische Chemie und Mikroskopie (10th ed.), Thieme-Verlag, Stuttgart, Germany (1966); R. J. Henry, Clinical Chemistry (1st ed.), Harper and Row, New York (1964); R. Richterich, Klinische Chemie (3rd ed.), Karger, Basel, Switzerland (1971); R. J. Henry et al.; Clinical Chemistry (2nd ed.), Harper and Row, New York (1974); and W. Rick, Klinische Chemie und Mikroskopie (4th ed.), Springer Verlag, Berlin/Heidelberg/New York (1976)], namely:
1. Spectrophotometric methods (as carboxy-hemoglobin, oxy-hemoglobin or cyano-hemiglobin (cyano-methemoglobin), as cyano-hematin, acid or alkaline hematin, as well as pyridine-hemochromogen);
2. Gasometric methods (determination of oxygen- or carbonmonoxide capacity);
3. Chemical methods (determination of iron content);
4. Other methods (refractometry, density measuring enzymatic reactions such as the pseudoperoxidase activity of the hemoglobin).
Among these, the spectrophotometric cyano-hemiglobin method has been universally accepted and is now being used worldwide. During the period from 1953 to 1963, after practically all of the laboratories had introduced this method in place of others [cf. J. Spaander, Die Verwertung von Blutuntersuchungen, Strahlenschutz in Forschung und Praxis 4, 122-127 (1964), and F. W. Sunderman, Status of Clinical Hemoglobinometry in the United States, Am. J. Clin. Pathol. 43, 9-15 (1965)] and a cyano-hemoglobin standard was recommended [R. K. Cannan, Proposal for a Certified Standard for Use in Hemoglobinometry--Second and Final Report, J. Lab. Clin. Med. 52, 471-476 (1958)], it was recommended for universal adoption [see Artzl. Labor. 8, 188 (1962); and Brit. J. Haemat. 13, 71-75 (1967)]. Since that time this method is recognized as the routine method.
In Germany it has acquired the function of a reference method, that is, other methods are allowed to be applied only if the relationship of the results obtained therewith to the result of the reference method is known [see German Norms: Determination of the hemoglobin content of blood; DIN Vornorm 58 931 (1970)].
The introduction of the cyano-hemiglobin method as a standard has engendered a certain bias against other methods among those skilled in the art: "Hemiglobin-cyanide is the only known stable hemoglobin-derivative" (R. Richterich, supra); "all other methods . . . are to be rejected" (W. Rick, supra); " . . . cyano-hemiglobin (most exact proof)" (L. Hallmann, supra).
The principle of the cyano-hemiglobin method consists of oxidizing the hemoglobin with potassium ferricyanide to hemiglobin and converting same with potassium cyanide to cyano-hemiglobin, using a modified so-called Drabkin's solution as the reaction solution, as indicated originally by van Kampen and Zijlstra [E. J. van Kampen and W. G. Zijlstra, Standardization of Hemoglobinometry, II; The Hemoglobincyanide Method. Clin. Chim. Acta 6, 538-544 (1961); and W. G. Zijlstra and E. J. van Kampen, Standardization of Hemoglobinometry, I. The Extinction Coefficient of Hemoglobincyanide at .eta.=540 m.mu.; .epsilon..sub.HiCN.sup.540, Clin. Chim. Acta 5, 719-726 (1960)]. This method has the following advantages:
1. Use of only one reaction solution;
2. All hemoglobin derivatives of the blood are included (desoxy-hemoglobin, oxy-hemoglobin, carboxy-hemoglobin, hemiglobin and substantially all sulf-hemoglobin as well);
3. Cyano-hemiglobin possesses a broad and flat extinction maximum at 540 nm, so that reliable results may be obtained even with unsophisticated filterphotometers.
4. The Lambert-Beer law is valid over a wide measuring range;
5. Production and shipment of stable standard solutions for control purposes is possible; they may be produced from crystalline hemoglobin as well as from washed erythrocytes;
6. Cyano-hemiglobin is a stable hemoglobin derivative, so that the measurement of the extinction can be effected after several minutes as well as several days later.
The disadvantages of the cyano-hemiglobin method are the following [R. J. Henry, Clinical Chemistry (1st ed.), page 740, Harper and Row, New York (1964)]:
1. The toxicity of the reaction solution requires special handling measures (use of mechanical pipettes, careful discarding of the solution in the sink);
2. The reaction solution does not remain stable over a long time; it is sensitive to light and must be stored correspondingly (brown flasks or the like);
3. The concentrations of the reactants have to be maintained exactly; this is especially applicable to the buffer, which has to guarantee a definite pH-value;
4. The various hemoglobin derivatives have different reaction periods, some of which are entirely too long. Originally, a reaction period of 3 minutes was indicated for all hemoglobin derivatives (E. J. van Kampen and W. G. Zijlstra, supra), but this statement had to be modified subsequently.
In 1965, J. B. Taylor and J. D. M. Miller [A source of error in the cyanmethemoglobin method of determination of hemoglobin concentration in blood containing carbon monoxide, Am. J. Clin. Pathol. 43, 265-271 (1965)] showed that the cyano-hemiglobin formation in the presence of carboxy-hemoglobin is distinctly prolonged and, therefore, considerable errors in the determination of hemoglobin must occur.
In 1965 E. J. van Kampen and W. G. Zijlstra [Advances in Clinical Chemistry, Vol. 8, page 160 (H. Sobotka and C. P. Stewart, editors), Academic Press, New York 1965] suggested a reaction time of 90 minutes in those instances where the blood to be tested is expected to contain carboxy-hemoglobin.
In 1967, F. L. Rodkey [Kinetic aspects of cyanmethemoglobin formation from carboxyhemoglobin, Clin. Chem. 13, 2-5 (1967)] showed that the conversion of HbCO into cyano-hemiglobin at room temperature takes 90 to 120 minutes, while HbO.sub.2 takes only 10 minutes. This author is able to shorten the reaction period for conversion of HbCO into cyano-hemiglobin to 15 minutes, if the concentration of potassium ferricyanide is increased five-fold. In 1967, E. W. Rice [Rapid determination of total hemoglobin as hemoglobin cyanide in blood containing carboxyhemoglobin, Clin. Chim. Acta 18, 89-91 (1967)] solved the problem by heating the solution to 56.degree. C. Then the reaction of HbCO to cyano-hemiglobin is complete after 3 to 5 minutes.
The fact that carboxy-hemoglobin oxidizes to hemiglobin much slower than other derivatives (Hb, HbO.sub.2), is utilized in the so-called Hoppe-Seyler test for a qualitative HbCO-proof in blood [cf. W. Massmann, Deutsche Med. Wochenschrift 79, 1140-1142 (1940)].
Further possibilities of error could be excluded. For example, a precipitation of plasma-proteins and a turbidity connected therewith could be avoided [cf. P. Green and C. F. J. Teal, Am. J. Clin. Pathol. 32, 216-217 (1959)] by adding a detergent to the reaction solution [E. J. van Kampen and W. G. Zijlstra, 1961, supra].